(W1130-11-62) Evaluation of Ice Nucleation/Freezing Phenomenon for a Monoclonal Antibody Drug Product Solution in Primary Container Closure Systems Using a Thermal Cycling Chamber

Purpose: Therapeutic monoclonal antibody (mAb) drug product (DP) solution intended for refrigerated storage (2-8°C) might exhibit ice nucleation/freezing due to temperature excursions below 0°C during shipping and distribution. Freezing of DP solution in its primary container closure system could compromise the integrity of the container-closure, increase the risk of microbial contamination, and/or increase the risk of freeze concentration gradient induced instability of the DP. Therefore, it's important not only to assess whether mAb DP will undergo ice nucleation and/or freezing at a given sub-zero temperature after prolonged hold duration, but also to establish sub-zero temperature excursion limits. The objective of this study was to assess the impact of protein concentration and primary container type on the formation of ice nucleation/freezing of a mAb DP when held at select subzero temperatures in the thermal cycling chamber for prolonged period.

Methods: The mAb DP solutions prepared at various protein concentrations (50 to 250 mg/mL) including control (buffer only), filtered through 0.2um membrane, and filled (~1.1 mL) into either ISO 2R glass vials/13mm butyl rubber stopper or in 1 mL glass pre-fillable syringes (PFS)/ butyl rubber plunger stopper using aseptic techniques under laminar air flow. The PFS and vials (n=10) were placed upright on the shelves in the thermal cycling chamber maintained at -5°C, -10°C, -15°C, or -20°C for up to 7 days at each temperature. The samples were inspected on each day visually for the formation of ice crystals or frozen solids. Additionally, a lyophilizer with controlled nucleation capability was utilized to assess the sub-zero temperature at which freezing could be forced for DP solutions in vials.

Results: Ice nucelation/freezing was not observed in all mAb DP and control samples in both vial and PFS when stored at -5°C for up to 7 days. In contrast, all samples froze instantly when forced to nucleate at -5°C shelf temperature using controlled lyo technology. At -10°C, mAb DP in PFS showed a greater propensity to freeze compared to mAb DP in vials. This may be due to the presence of silicone oil in the syringe acting as a nucleation inducer. Majority of samples showed freezing when storage temperature was -15°C with all samples froze instantly at -20°C irrespective of protein concentrations and container types studied.

Conclusion: The results showed low risk of freezing of mAb DP solution when exposed to short storage durations at -5°C irrespective of protein concentrations and container types studied. At and below -10⁰C, the risk of DP solution freezing increases particularly in PFS. Under forced controlled nucleation conditions, regardless of protein concentration (up to 250 mg/mL), mAb DP solutions in vials can be induced to freeze even at -5⁰C. The study furthers our understanding of the risks of ice nucleation/freezing in mAb DP solution in primary container closure systems when exposed to short term temperature excursions and helps to design thermal cycling study conditions.